Solid forms of an antiviral compound

ABSTRACT

Crystalline solid forms and the amorphous form of the anti-HCV compound 5-(3,3-dimethylbutyn-1-yl)-3-[(cis-4-hydroxy-4-{[(3S)-tetrahydrofuran-3-yloxy]methyl}cyclohexyl){[(1R)-4-methylcyclohex-3-en-1-yl]carbonyl}amino]thiophene-2-carboxylic acid (Compound I) were prepared and characterized in the solid state: 
                         
Also provided are processes of manufacture and methods of using the crystalline forms.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. §119(e) to U.S.Provisional Application Ser. No. 61/684,297, filed on Aug. 17, 2012, theentirety of which is incorporated herein by reference.

BACKGROUND

The present disclosure relates generally to crystalline solid forms andamorphous solid form of the antiviral compound5-(3,3-dimethylbutyn-1-yl)-3-[(cis-4-hydroxy-4-{[(3S)-tetrahydrofuran-3-yloxy]methyl}cyclohexyl){[(1R)-4-methylcyclohex-3-en-1-yl]carbonyl}amino]thiophene-2-carboxylicacid, processes for making the forms, and their therapeutic methods ofuse.

Hepatitis C is recognized as a chronic viral disease of the liver whichis characterized by liver disease. Although drugs targeting the liverare in wide use and have shown effectiveness, toxicity and other sideeffects have limited their usefulness Inhibitors of hepatitis C virus(HCV) are useful to limit the establishment and progression of infectionby HCV as well as in diagnostic assays for HCV.

The compound5-(3,3-dimethylbutyn-1-yl)-3-[(cis-4-hydroxy-4-{[(3S)-tetrahydrofuran-3-yloxy]methyl}cyclohexyl){[(1R)-4-methylcyclohex-3-en-1-yl]carbonyl}amino]thiophene-2-carboxylicacid, designated herein as Compound I, is known to be an effectiveanti-HCV agent, as described for example in WO 2011/088345. However,Compound I was not heretofore known in any crystalline or amorphoussolid form.

SUMMARY

The present disclosure fulfills these needs and others by providingamorphous and crystalline forms of Compound I, hydrates and solvates ofCompound I. The disclosure also provides processes for making thecrystalline forms and methods for using them in the treatment of HCV.Specifically, twelve crystalline solid forms of Compound I have beendiscovered through various screening techniques. Form II is an anhydrouscrystalline form as it is stable and non-hygroscopic under ambientconditions. Other forms are hydrates, solvates, or desolvated forms. Inaddition to the crystalline forms described below, in one embodiment,the amorphous solid form is also provided.

One embodiment is crystalline5-(3,3-dimethylbutyn-1-yl)-3-[(cis-4-hydroxy-4-{[(3S)-tetrahydrofuran-3-yloxy]methyl}cyclohexyl){[(1R)-4-methylcyclohex-3-en-1-yl]carbonyl}amino]thiophene-2-carboxylicacid hydrate (Compound I Form I) characterized by an X-ray powderdiffractogram comprising at least three of the following peaks: 4.0,7.3, 8.1, 17.9, or 21.8 °2θ±0.2 °2θ, as determined on a diffractometerusing Cu-Kαradiation.

Another embodiment is crystalline5-(3,3-dimethylbutyn-1-yl)-3-[(cis-4-hydroxy-4-{[(3S)-tetrahydrofuran-3-yloxy]methyl}cyclohexyl){[(1R)-4-methylcyclohex-3-en-1-yl]carbonyl}amino]thiophene-2-carboxylicacid (Compound I Form II) characterized by an X-ray powder diffractogramcomprising at least three of the following peaks: 5.5, 6.0, 16.9, 18.1,or 21.3 °2θ±0.2 °2θ, as determined on a diffractometer using Cu-Kα.

Another embodiment is crystalline5-(3,3-dimethylbutyn-1-yl)-3-[(cis-4-hydroxy-4-{[(3S)-tetrahydrofuran-3-yloxy]methyl}cyclohexyl){[(1R)-4-methylcyclohex-3-en-1-yl]carbonyl}amino]thiophene-2-carboxylicacid hydrate and/or solvate (Compound I Form III) characterized by anX-ray powder diffractogram comprising at least three of the followingpeaks: 4.1, 6.8, 8.9, 17.4, or 21.4 °2θ±0.2 °2θ, as determined on adiffractometer using Cu-Kα.

Another embodiment is crystalline5-(3,3-dimethylbutyn-1-yl)-3-[(cis-4-hydroxy-4-{[(3S)-tetrahydrofuran-3-yloxy]methyl}cyclohexyl){[(1R)-4-methylcyclohex-3-en-1-yl]carbonyl}amino]thiophene-2-carboxylicacid hydrate and/or solvate (Compound I Form IV), characterized by anX-ray powder diffractogram comprising at least three of the followingpeaks: 4.1, 7.0, 9.0, 17.7, and 21.8 °2θ±0.2 °2θ, as determined on adiffractometer using Cu-Kα.

Another embodiment is crystalline5-(3,3-dimethylbutyn-1-yl)-3-[(cis-4-hydroxy-4-{[(3S)-tetrahydrofuran-3-yloxy]methyl}cyclohexyl){[(1R)-4-methylcyclohex-3-en-1-yl]carbonyl}amino]thiophene-2-carboxylicacid solvate (Compound I Form V), characterized by an X-ray powderdiffractogram comprising at least three of the following peaks: 4.2,7.0, 7.8, 8.4, or 21.1 °2θ±0.2 °2θ, as determined on a diffractometerusing Cu-Kα.

In one embodiment the solvate is selected from the group consisting ofmethyl tert-butyl ether (MTBE) solvate (Compound I Form V-MTBE),isopropyl alcohol (IPA) solvate (Compound I Form V-IPA), ethanol (EtOH)solvate (Compound I Form V-EtOH), methyl ethyl ketone solvate (MEK)solvate (Compound I Form V-MEK), and 2-methyl tetrahydrofuran (2-Me-THF)solvate (Compound I Form V-2-Me-THF).

Another embodiment is crystalline5-(3,3-dimethylbutyn-1-yl)-3-[(cis-4-hydroxy-4-{[(3S)-tetrahydrofuran-3-yloxy]methyl}cyclohexyl){[(1R)-4-methylcyclohex-3-en-1-yl]carbonyl}amino]thiophene-2-carboxylicacid diisopropyl ether solvate (Compound I Form VI), characterized by anX-ray powder diffractogram comprising at least three of the followingpeaks: 5.3, 7.0, 8.1, 17.2, 19.0 °2θ±0.2 °2θ, as determined on adiffractometer using Cu-Kα.

Another embodiment is crystalline5-(3,3-dimethylbutyn-1-yl)-3-[(cis-4-hydroxy-4-{[(3S)-tetrahydrofuran-3-yloxy]methyl}cyclohexyl){[(1R)-4-methylcyclohex-3-en-1-yl]carbonyl}amino]thiophene-2-carboxylicacid isopropyl alcohol solvate (Compound I Form VII), characterized byan X-ray powder diffractogram comprising at least three of the followingpeaks: 4.6, 5.9, 8.8, 9.3, or 21.5 °2θ±0.2 °2θ, as determined on adiffractometer using Cu-Kα.

Another embodiment is crystalline5-(3,3-dimethylbutyn-1-yl)-3-[(cis-4-hydroxy-4-{[(3S)-tetrahydrofuran-3-yloxy]methyl}cyclohexyl){[(1R)-4-methylcyclohex-3-en-1-yl]carbonyl}amino]thiophene-2-carboxylicacid monohydrate (Compound I Form VIII), characterized by an X-raypowder diffractogram comprising at least three of the following peaks:3.8, 7.7, 7.9, 18.0, or 21.7 °2θ±0.2 °2θ, as determined on adiffractometer using Cu-Kα.

Another embodiment is crystalline5-(3,3-dimethylbutyn-1-yl)-3-[(cis-4-hydroxy-4-{[(3S)-tetrahydrofuran-3-yloxy]methyl}cyclohexyl){[(1R)-4-methylcyclohex-3-en-1-yl]carbonyl}amino]thiophene-2-carboxylicacid ethanol solvate (Compound I Form IX), characterized by an X-raypowder diffractogram comprising at least three of the following peaks:4.3, 7.0, 7.7, 8.5, or 16.9 °2θ±0.2 °2θ, as determined on adiffractometer using Cu-Kα.

Another embodiment is crystalline5-(3,3-dimethylbutyn-1-yl)-3-[(cis-4-hydroxy-4-{[(3S)-tetrahydrofuran-3-yloxy]methyl}cyclohexyl){[(1R)-4-methylcyclohex-3-en-1-yl]carbonyl}amino]thiophene-2-carboxylicacid methyl ethyl ketone (Compound I Form X), characterized by an X-raypowder diffractogram comprising at least three of the following peaks:6.9, 8.0, 15.7, 16.1, or 17.5 °2θ±0.2 °2θ, as determined on adiffractometer using Cu-Kα.

Another embodiment is crystalline5-(3,3-dimethylbutyn-1-yl)-3-[(cis-4-hydroxy-4-{[(3S)-tetrahydrofuran-3-yloxy]methyl}cyclohexyl){[(1R)-4-methylcyclohex-3-en-1-yl]carbonyl}amino]thiophene-2-carboxylicacid (Compound I Form XI), characterized by an X-ray powderdiffractogram comprising at least three of the following peaks: 5.4,5.7, 7.0, 7.9, or 8.6 °2θ±0.2 °2θ, as determined on a diffractometerusing Cu-Kα.

Another embodiment is crystalline5-(3,3-dimethylbutyn-1-yl)-3-[(cis-4-hydroxy-4-{[(3S)-tetrahydrofuran-3-yloxy]methyl}cyclohexyl){[(1R)-4-methylcyclohex-3-en-1-yl]carbonyl}amino]thiophene-2-carboxylicacid ethanol solvate (Compound I Form XII), characterized by an X-raypowder diffractogram comprising at least three of the following peaks:4.5, 6.4, 7.1, 8.5, or 8.9 °2θ±0.2 °2θ, as determined on adiffractometer using Cu-Kα.

One embodiment is directed to a method for treating a subject sufferingfrom hepatitis C virus (HCV), comprising administering to the subject atherapeutically effective amount of a compound as described throughout.

In another embodiment is provided a pharmaceutical compositioncomprising a compound as described throughout and a pharmaceuticallyacceptable excipient.

In one embodiment is a process for making Compound I Form I, comprisingcontacting a5-(3,3-dimethylbutyn-1-yl)-3-[(cis-4-hydroxy-4-{[(3S)-tetrahydrofuran-3-yloxy]methyl}cyclohexyl){[(1R)-4-methylcyclohex-3-en-1-yl]carbonyl}amino]thiophene-2-carboxylicacid with water and a solvent selected from the group consisting ofacetonitrile, methanol, and acetone, whereby Compound I Form I isformed.

In another embodiment is a process for making Compound I Form II,comprising contacting5-(3,3-dimethylbutyn-1-yl)-3-[(cis-4-hydroxy-4-{[(3S)-tetrahydrofuran-3-yloxy]methyl}cyclohexyl){[(1R)-4-methylcyclohex-3-en-1-yl]carbonyl}amino]thiophene-2-carboxylicacid with acetonitrile, whereby Compound I Form II is formed.

In another embodiment is a process for making Compound I Form III,comprising contacting5-(3,3-dimethylbutyn-1-yl)-3-[(cis-4-hydroxy-4-{[(3S)-tetrahydrofuran-3-yloxy]methyl}cyclohexyl){[(1R)-4-methylcyclohex-3-en-1-yl]carbonyl}amino]thiophene-2-carboxylicacid with ethanol and water, whereby Compound I Form III is formed.

In another embodiment is a process for making Compound I Form IV,comprising contacting5-(3,3-dimethylbutyn-1-yl)-3-[(cis-4-hydroxy-4-{[(3S)-tetrahydrofuran-3-yloxy]methyl}cyclohexyl){[(1R)-4-methylcyclohex-3-en-1-yl]carbonyl}amino]thiophene-2-carboxylicacid with ethanol and water, whereby Compound I Form IV is formed.

In another embodiment is a process for making Compound I Form V-MTBE,Compound I Form V-IPA, Compound I Form V-EtOH, Compound I Form V-MEK, orCompound I Form V-2-Me-THF, comprising contacting5-(3,3-dimethylbutyn-1-yl)-3-[(cis-4-hydroxy-4-{[(3S)-tetrahydrofuran-3-yloxy]methyl}cyclohexyl){[(1R)-4-methylcyclohex-3-en-1-yl]carbonyl}amino]thiophene-2-carboxylicacid with a solvent or solvent mixture selected from the groupconsisting of methyl tert-butyl ether, isopropyl alcohol and watermixture, ethanol and water mixture, methyl ethyl ketone and heptanemixture, and 2-methyl tetrahydrofuran and heptane mixture, wherebyCompound I Form V-MTBE, Compound I Form V-IPA, Compound I Form V-EtOH,Compound I Form V-MEK, or Compound I Form V-2-Me-THF, respectively, isformed.

In another embodiment is a process for making Compound I Form VI,comprising contacting5-(3,3-dimethylbutyn-1-yl)-3-[(cis-4-hydroxy-4-{[(3S)-tetrahydrofuran-3-yloxy]methyl}cyclohexyl){[(1R)-4-methylcyclohex-3-en-1-yl]carbonyl}amino]thiophene-2-carboxylicacid with diisopropyl ether, whereby Compound I Form VI is formed.

In another embodiment is a process for making Compound I Form VII,comprising contacting5-(3,3-dimethylbutyn-1-yl)-3-[(cis-4-hydroxy-4-{[(3S)-tetrahydrofuran-3-yloxy]methyl}cyclohexyl){[(1R)-4-methylcyclohex-3-en-1-yl]carbonyl}amino]thiophene-2-carboxylicacid with isopropyl alcohol, whereby Compound I Form VII is formed.

In another embodiment is a process for making Compound I Form VIII,comprising contacting5-(3,3-dimethylbutyn-1-yl)-3-[(cis-4-hydroxy-4-{[(3S)-tetrahydrofuran-3-yloxy]methyl}cyclohexyl){[(1R)-4-methylcyclohex-3-en-1-yl]carbonyl}amino]thiophene-2-carboxylicacid with ethanol and water, whereby Compound I Form VIII is formed.

In another embodiment is a process for making Compound I Form IX,comprising contacting5-(3,3-dimethylbutyn-1-yl)-3-[(cis-4-hydroxy-4-{[(3S)-tetrahydrofuran-3-yloxy]methyl}cyclohexyl){[(1R)-4-methylcyclohex-3-en-1-yl]carbonyl}amino]thiophene-2-carboxylicacid with ethanol, whereby Compound I Form IX is formed.

In another embodiment is a process for making Compound I Form X,comprising contacting5-(3,3-dimethylbutyn-1-yl)-3-[(cis-4-hydroxy-4-{[(3S)-tetrahydrofuran-3-yloxy]methyl}cyclohexyl){[(1R)-4-methylcyclohex-3-en-1-yl]carbonyl}amino]thiophene-2-carboxylicacid with methyl ethyl ketone, whereby Compound I Form X is formed.

In another embodiment is a process for making Compound I Form XI,comprising desolvating Compound I Form X, whereby Compound I Form XI isformed.

In another embodiment is a process for making Compound I Form XII,comprising contacting Compound I Form I with ethanol in water, wherebyCompound I Form XII is formed.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an X-ray powder diffraction pattern of Compound I Form I.

FIG. 2 is a combined differential scanning calorimetry plot (DSC; bottomcurve) and thermogravimetric analysis (TGA; top curve) of Compound IForm I.

FIG. 3 provides X-ray powder diffraction patterns of Form I obtainedfrom ACN/water, MeOH/water, and acetone/water mixtures.

FIG. 4 is an X-ray powder diffraction pattern of Compound I Form II.

FIG. 5 is a combined differential scanning calorimetry plot (DSC; bottomcurve) and thermogravimetric analysis (TGA; top curve) of Compound IForm II.

FIG. 6 is an X-ray powder diffraction pattern showing that Form IIconverts slowly to Form I in water at 48° C.

FIG. 7 are X-ray powder diffraction patterns of Form I solids after itwas dried at 50° C. and 70° C.

FIG. 8 is an X-ray powder diffraction pattern of Compound I Form III.

FIG. 9 is a combined differential scanning calorimetry plot (DSC; bottomcurve) and thermogravimetric analysis (TGA; top curve) of Compound IForm III.

FIG. 10 is an X-ray powder diffraction pattern of Compound I Form IV.

FIG. 11 is a combined differential scanning calorimetry plot (DSC; topcurve) and thermogravimetric analysis (TGA; bottom curve) of Compound IForm IV.

FIG. 12 is an X-ray powder diffraction pattern of Compound I Form V,obtained from MTBE, IPA/water, MEK/heptane, 2-Me-THF/heptane.

FIG. 13 is a combined differential scanning calorimetry plot (DSC; topcurve) and thermogravimetric analysis (TGA; bottom curve) of Compound IForm V.

FIG. 14 is an X-ray powder diffraction pattern of Compound I Form VI.

FIG. 15 is a combined differential scanning calorimetry plot (DSC;bottom curve) and thermogravimetric analysis (TGA; top curve) ofCompound I Form VI.

FIG. 16 is an X-ray powder diffraction pattern of Compound I Form VII.

FIG. 17 is a combined differential scanning calorimetry plot (DSC;bottom curve) and thermogravimetric analysis (TGA; top curve) ofCompound I Form VII.

FIG. 18 is an X-ray powder diffraction pattern of Compound I Form VIII.

FIG. 19 is a combined differential scanning calorimetry plot (DSC;bottom curve) and thermogravimetric analysis (TGA; top curve) ofCompound I Form VIII

FIG. 20 is an X-ray powder diffraction pattern of Compound I Form IX.

FIG. 21 is a combined differential scanning calorimetry plot (DSC; topcurve) and thermogravimetric analysis (TGA; bottom curve) of Compound IForm IX

FIG. 22 is an X-ray powder diffraction pattern of Compound I Form X.

FIG. 23 is a combined differential scanning calorimetry plot (DSC; topcurve) and thermogravimetric analysis (TGA; bottom curve) of Compound IForm X.

FIG. 24 is an X-ray powder diffraction pattern of Compound I Form XI.

FIG. 25 is a thermogravimetric analysis (TGA) of Compound I Form XI.

FIG. 26 is an X-ray powder diffraction pattern of Compound I Form XII.

FIG. 27 is a thermogravimetric analysis (TGA) of Compound I Form XII.

FIG. 28 is an X-ray powder diffraction pattern of amorphous Compound I.

DETAILED DESCRIPTION

The compound5-(3,3-dimethylbutyn-1-yl)-3-[(cis-4-hydroxy-4-{[(3S)-tetrahydrofuran-3-yloxy]methyl}cyclohexyl){[(1R)-4-methylcyclohex-3-en-1-yl]carbonyl}amino]thiophene-2-carboxylicacid (Compound I) is a selective and potent inhibitor of HCV NS5B.

The present invention results from the surprising discoveries of theamorphous form and crystalline forms of Compound I, advantagesattributed to the forms as described herein, and processes for makingthe crystalline forms. For example Form II is particularly desirable asit is contemplated to be stable during a wet granulation. Further, it iscontemplated that the amorphous form has improved bioavailability.

DEFINITIONS

As used in the present specification, the following words and phrasesare generally intended to have the meanings as set forth below, exceptto the extent that the context in which they are used indicatesotherwise.

The term “hydrate” refers to a complex formed by the combining ofCompound I and water. The term includes hemihydrates and channelhydrates.

The term “solvate” refers to a complex formed by the combining ofCompound I and a solvent.

The term “desolvated” refers to a Compound I form that is a solvate asdescribed herein, and from which solvent molecules have been partiallyor completely removed. Desolvation techniques to produce desolvatedforms include, without limitation, exposure of a Compound I Form(solvate) to a vacuum, subjecting the solvate to elevated temperature,exposing the solvate to a stream of gas, such as air or nitrogen, or anycombination thereof. Thus, a desolvated Compound I form can beanhydrous, i.e., completely without solvent molecules, or partiallysolvated wherein solvent molecules are present in stoichiometric ornon-stoichiometric amounts.

Any formula or structure given herein, including Compound I, is alsointended to represent unlabeled forms as well as isotopically labeledforms of the compounds. Isotopically labeled compounds have structuresdepicted by the Formulae given herein except that one or more atoms arereplaced by an atom having a selected atomic mass or mass number.Examples of isotopes that can be incorporated into compounds of thedisclosure include isotopes of hydrogen, carbon, nitrogen, oxygen,phosphorous, fluorine and chlorine, such as, but not limited to ²H(deuterium D), ³H (tritium), ¹¹C, ¹³C, ¹⁴C, ¹⁵N, ¹⁸F, ³¹P, ³²P, ³⁵S,³⁶Cl and ¹²⁵I. Various isotopically labeled compounds of the presentdisclosure, for example those into which radioactive isotopes such as³H, ¹³C and ¹⁴C are incorporated. Such isotopically labeled compoundsmay be useful in metabolic studies, reaction kinetic studies, detectionor imaging techniques, such as positron emission tomography (PET) orsingle-photon emission computed tomography (SPECT) including drug orsubstrate tissue distribution assays or in radioactive treatment ofpatients.

The disclosure also includes Compound I in which from 1 to “n” hydrogensattached to a carbon atom is/are replaced by deuterium, in which n isthe number of hydrogens in the molecule. Such compounds exhibitincreased resistance to metabolism and are thus useful for increasingthe half life of any Compound I when administered to a mammal. See, forexample, Foster, “Deuterium Isotope Effects in Studies of DrugMetabolism”, Trends Pharmacol. Sci. 5(12):524-527 (1984). Such compoundsare synthesized by means well known in the art, for example by employingstarting materials in which one or more hydrogen atoms have beenreplaced by deuterium.

Deuterium labeled or substituted therapeutic compounds of the disclosuremay have improved DMPK (drug metabolism and pharmacokinetics)properties, relating to distribution, metabolism and excretion (ADME).Substitution with heavier isotopes such as deuterium may afford certaintherapeutic advantages resulting from greater metabolic stability, forexample increased in vivo half-life or reduced dosage requirements. An¹⁸F labeled compound may be useful for PET or SPECT studies.Isotopically labeled compounds of this disclosure and prodrugs thereofcan generally be prepared by carrying out the procedures disclosed inthe schemes or in the examples and preparations described below bysubstituting a readily available isotopically labeled reagent for anon-isotopically labeled reagent. Further, substitution with heavierisotopes, particularly deuterium (i.e., ²H or D) may afford certaintherapeutic advantages resulting from greater metabolic stability, forexample increased in vivo half-life or reduced dosage requirements or animprovement in therapeutic index. It is understood that deuterium inthis context is regarded as a substituent in Compound I.

The concentration of such a heavier isotope, specifically deuterium, maybe defined by an isotopic enrichment factor. In the compounds of thisdisclosure any atom not specifically designated as a particular isotopeis meant to represent any stable isotope of that atom. Unless otherwisestated, when a position is designated specifically as “H” or “hydrogen”,the position is understood to have hydrogen at its natural abundanceisotopic composition. Accordingly, in the compounds of this disclosureany atom specifically designated as a deuterium (D) is meant torepresent deuterium.

The term “therapeutically effective amount” refers to an amount that issufficient to effect treatment, as defined below, when administered to amammal in need of such treatment. The therapeutically effective amountwill vary depending upon the subject being treated, the weight and ageof the subject, the severity of the disease condition, the manner ofadministration and the like, which can readily be determined by one ofordinary skill in the art.

In addition, abbreviations as used herein have respective meanings asfollows:

2-Me THF 2 methyl tetrahydrofuran Ac Acetate ACN Acetonitrile BippyPhos5-(di-tert-butylphosphino)-1′,3′,5′- triphenyl-1′H-[1,4′]bipyrazole BnBenzyl br. s Broad singlet Bu Butyl dba Dibenzylideneacetone DCMDichloromethane dd Doublet of doublets ddd Doublet of doublet ofdoublets DIPE diisopropyl ether DMF Dimethylformamide DMSODimethylsulfoxide dr Diastereomeric ratio DSC Differential scanningcalorimetry DVS Dynamic vapor sorption ee Enantiomeric excess equivEquivalents Et Ethyl EtOAc Ethyl acetate EtOH Ethanol ft Foot (length) gGram GC Gas chromatography h Hour HCV Hepatitis C virus HPLCHigh-pressure liquid chromatography IPA Isopropyl alcohol IPAc Isopropylacetate iPr Isopropyl iPrOAc isopropyl acetate kg Kilogram L Liter mMultiplet M Molar Me Methyl MeCl₂ methylene chloride MEK methyl ethylketone MeOH methanol mg Milligram MHz Mega hertz MIBK Methylisobutylketone min Minute mL Milliliter mmol Millimole mol Mole MTBEMethyl-tert-butyl ether N Normal NLT No less than NMR Nuclear magneticresonance Ph Phenyl RH Relative humidity s Singlet t-Bu tert-Butyl tdTriplet of doublets Tf Trifluoromethanesulfonate TGA Thermogravimetricanalysis THF Tetrahydrofuran TMS Trimethylsilyl vol Volume wt WeightXRPD X-ray powder diffraction δ Chemical shift μL Microliter

Solid Forms of Compound I

One embodiment is crystalline5-(3,3-dimethylbutyn-1-yl)-3-[(cis-4-hydroxy-4-{[(3S)-tetrahydrofuran-3-yloxy]methyl}cyclohexyl){[(1R)-4-methylcyclohex-3-en-1-yl]carbonyl}amino]thiophene-2-carboxylicacid hydrate (Compound I Form I) characterized by an X-ray powderdiffractogram comprising at least three of the following peaks: 4.0,7.3, 8.1, 17.9, or 21.8 °2θ±0.2 °2θ, as determined on a diffractometerusing Cu-Kαradiation. In another embodiment, the diffractogram comprisespeaks at 4.0, 7.3, 8.1, 17.9, and 21.8 °2θ±0.2 °2θ. In anotherembodiment, the diffractogram is substantially as shown in FIG. 1.

In one embodiment, Compound I Form I is characterized by a differentialscanning calorimetry (DSC) curve that comprises two endotherms at about55° C. to about 115° C. and about 127° C. In another embodiment, the DSCcurve is substantially as shown in FIG. 2.

In one embodiment, Compound I Form I is characterized by a weight loss,as measured by thermogravimetric analysis (TGA), of about 5.4% at about95° C. In one embodiment, the TGA is substantially as shown in FIG. 2.

Another embodiment is crystalline5-(3,3-dimethylbutyn-1-yl)-3-[(cis-4-hydroxy-4-{[(3S)-tetrahydrofuran-3-yloxy]methyl}cyclohexyl){[(1R)-4-methylcyclohex-3-en-1-yl]carbonyl}amino]thiophene-2-carboxylicacid (Compound I Form II) characterized by an X-ray powder diffractogramcomprising at least three of the following peaks: 5.5, 6.0, 16.9, 18.1,or 21.3 °2θ±0.2 °2θ, as determined on a diffractometer using Cu-Kα. Inone embodiment, the diffractogram comprises peaks at 5.5, 6.0, 16.9,18.1, and 21.3 °2θ±0.2 °2θ. In one embodiment, the diffractogram issubstantially as shown in FIG. 4.

In one embodiment, Compound I Form II is characterized by a differentialscanning calorimetry (DSC) curve that comprises an endotherm at about162° C. In another embodiment, the DSC curve is substantially as shownin FIG. 5.

In one embodiment, Compound I Form II is characterized by a weight loss,as measured by thermogravimetric analysis (TGA), of about 0.2% at about120° C. In one embodiment, the TGA is substantially as shown in FIG. 5.

Another embodiment is crystalline5-(3,3-dimethylbutyn-1-yl)-3-[(cis-4-hydroxy-4-{[(3S)-tetrahydrofuran-3-yloxy]methyl}cyclohexyl){[(1R)-4-methylcyclohex-3-en-1-yl]carbonyl}amino]thiophene-2-carboxylicacid hydrate and/or solvate (Compound I Form III) characterized by anX-ray powder diffractogram comprising at least three of the followingpeaks: 4.1, 6.8, 8.9, 17.4, or 21.4 °2θ±0.2 °2θ, as determined on adiffractometer using Cu-Kα. In one embodiment, the compound is ahydrate. In one embodiment, the compound is an ethanol solvate (CompoundI Form III-EtOH). In one embodiment, the diffractogram comprises peaksat 4.1, 6.8, 8.9, 17.4, and 21.4 °2θ±0.2 °2θ. In one embodiment, thediffractogram is substantially as shown in FIG. 8.

In one embodiment, Compound I Form III is characterized by adifferential scanning calorimetry (DSC) curve that comprises twoendotherms at about 80° C. to about 110° C. and about 135° C. In oneembodiment, the DSC curve is substantially as shown in FIG. 9.

In one embodiment, Compound I Form III is characterized by two weightloss events, as measured by thermogravimetric analysis (TGA), of about1.6% at about 70° C. and 2.8% at about 150° C. In one embodiment, theTGA is substantially as shown in FIG. 9.

Another embodiment is crystalline5-(3,3-dimethylbutyn-1-yl)-3-[(cis-4-hydroxy-4-{[(3S)-tetrahydrofuran-3-yloxy]methyl}cyclohexyl){[(1R)-4-methylcyclohex-3-en-1-yl]carbonyl}amino]thiophene-2-carboxylicacid hydrate and/or solvate (Compound I Form IV), characterized by anX-ray powder diffractogram comprising at least three of the followingpeaks: 4.1, 7.0, 9.0, 17.7, and 21.8 °2θ±0.2 °2θ, as determined on adiffractometer using Cu-Kα. In one embodiment, the compound is ahydrate. In one embodiment, the compound is an ethanol solvate (CompoundI Form IV-EtOH). In one embodiment, the diffractogram comprises peaks at4.1, 7.0, 9.0, 17.7, and 21.8 °2θ±0.2 °2θ. In one embodiment, thediffractogram is substantially as shown in FIG. 10.

In one embodiment, Compound I Form IV is characterized by a differentialscanning calorimetry (DSC) curve that comprises two endotherms at about78 to about 110° C. and about 133° C. In one embodiment, the DSC curveis substantially as shown in FIG. 11.

In one embodiment, Compound I Form IV is characterized by two weightloss events, as measured by thermogravimetric analysis (TGA), of about1.2% at about 50° C. and 1.6% at about 100° C. In one embodiment, theTGA is substantially as shown in FIG. 11.

Another embodiment is crystalline5-(3,3-dimethylbutyn-1-yl)-3-[(cis-4-hydroxy-4-{[(3S)-tetrahydrofuran-3-yloxy]methyl}cyclohexyl){[(1R)-4-methylcyclohex-3-en-1-yl]carbonyl}amino]thiophene-2-carboxylicacid solvate (Compound I Form V), characterized by an X-ray powderdiffractogram comprising at least three of the following peaks: 4.2,7.0, 7.8, 8.4, or 21.1 °2θ±0.2 °2θ, as determined on a diffractometerusing Cu-Kα. The solvate is selected from the group consisting of methyltert-butyl ether (MTBE) solvate (Compound I Form V-MTBE), isopropylalcohol (IPA) solvate (Compound I Form V-IPA), ethanol (EtOH) solvate(Compound I Form V-EtOH), methyl ethyl ketone solvate (MEK) solvate(Compound I Form V-MEK), and 2-methyl tetrahydrofuran (2-Me-THF) solvate(Compound I Form V-2-Me-THF). In one embodiment, the diffractogramcomprises peaks at 4.2, 7.0, 7.8, 8.4, and 21.1 °2θ±0.2 °2θ. In oneembodiment, the diffractogram is substantially as shown in FIG. 12.

In one embodiment, Compound I Form V-EtOH is characterized by adifferential scanning calorimetry (DSC) curve that comprises anendotherm at about 133° C. to about 141° C. In one embodiment, the DSCcurve is substantially as shown in FIG. 13.

In one embodiment, Compound I Form V is characterized by a weight loss,as measured by thermogravimetric analysis (TGA), of about 4.8% at about120° C. for Compound I Form V-MTBE, 4.2% at about 130° C. for Compound IForm V-IPA, and 3.5% at about 140° C. for Compound I Form V-EtOH. In oneembodiment, the TGA is substantially as shown in FIG. 13.

Another embodiment is crystalline5-(3,3-dimethylbutyn-1-yl)-3-[(cis-4-hydroxy-4-{[(3S)-tetrahydrofuran-3-yloxy]methyl}cyclohexyl){[(1R)-4-methylcyclohex-3-en-1-yl]carbonyl}amino]thiophene-2-carboxylicacid diisopropyl ether solvate (Compound I Form VI), characterized by anX-ray powder diffractogram comprising at least three of the followingpeaks: 5.3, 7.0, 8.1, 17.2, 19.0 °2θ±0.2 °2θ, as determined on adiffractometer using Cu-Kα. In one embodiment, the diffractogramcomprises peaks at 5.3, 7.0, 8.1, 17.2, and 19.0 °2θ±0.2 °2θ. In oneembodiment, the diffractogram is substantially as shown in FIG. 14.

In one embodiment, Compound I Form VI is characterized by a differentialscanning calorimetry (DSC) curve that comprises an endotherm at about147° C. In one embodiment, the DSC curve is substantially as shown inFIG. 15.

In one embodiment, Compound I Form VI is characterized by a weight loss,as measured by thermogravimetric analysis (TGA), of about 4.6% at about150° C. In one embodiment, the TGA is substantially as shown in FIG. 15.

Another embodiment is crystalline5-(3,3-dimethylbutyn-1-yl)-3-[(cis-4-hydroxy-4-{[(3S)-tetrahydrofuran-3-yloxy]methyl}cyclohexyl){[(1R)-4-methylcyclohex-3-en-1-yl]carbonyl}amino]thiophene-2-carboxylicacid isopropyl alcohol solvate (Compound I Form VII), characterized byan X-ray powder diffractogram comprising at least three of the followingpeaks: 4.6, 5.9, 8.8, 9.3, or 21.5 °2θ±0.2 °2θ, as determined on adiffractometer using Cu-Kα.

In one embodiment, the diffractogram comprises peaks at 4.6, 5.9, 8.8,9.3, and 21.5 °2θ±0.2 °2θ. In one embodiment, the diffractogram issubstantially as shown in FIG. 16.

In one embodiment, Compound I Form VII is characterized by adifferential scanning calorimetry (DSC) curve that comprises anendotherm at about 128° C. In one embodiment, the DSC curve issubstantially as shown in FIG. 17.

In one embodiment, Compound I Form VII is characterized by a weightloss, as measured by thermogravimetric analysis (TGA), of about 11.3% atabout 140° C. In one embodiment, the TGA is substantially as shown inFIG. 17.

Another embodiment is crystalline5-(3,3-dimethylbutyn-1-yl)-3-[(cis-4-hydroxy-4-{[(3S)-tetrahydrofuran-3-yloxy]methyl}cyclohexyl){[(1R)-4-methylcyclohex-3-en-1-yl]carbonyl}amino]thiophene-2-carboxylicacid monohydrate (Compound I Form VIII), characterized by an X-raypowder diffractogram comprising at least three of the following peaks:3.8, 7.7, 7.9, 18.0, or 21.7 °2θ±0.2 °2θ, as determined on adiffractometer using Cu-Kα. In one embodiment, the diffractogramcomprises peaks at 3.8, 7.7, 7.9, 18.0, and 21.7 °2θ±0.2 °2θ. In oneembodiment, the diffractogram is substantially as shown in FIG. 18.

In one embodiment, Compound I Form VIII is characterized by adifferential scanning calorimetry (DSC) curve that comprises anendotherm at about 144° C. In one embodiment, the DSC curve issubstantially as shown in FIG. 19.

In one embodiment, Compound I Form VIII is characterized by a weightloss, as measured by thermogravimetric analysis (TGA), of about 3.2% atabout 100° C. In one embodiment, the TGA is substantially as shown inFIG. 19.

Another embodiment is crystalline5-(3,3-dimethylbutyn-1-yl)-3-[(cis-4-hydroxy-4-{[(3S)-tetrahydrofuran-3-yloxy]methyl}cyclohexyl){[(1R)-4-methylcyclohex-3-en-1-yl]carbonyl}amino]thiophene-2-carboxylicacid ethanol solvate (Compound I Form IX), characterized by an X-raypowder diffractogram comprising at least three of the following peaks:4.3, 7.0, 7.7, 8.5, or 16.9 °2θ±0.2 °2θ, as determined on adiffractometer using Cu-Kα. In one embodiment, the diffractogramcomprises peaks at 4.3, 7.0, 7.7, 8.5, and 16.9 °2θ±0.2 °2θ. In oneembodiment, the diffractogram is substantially as shown in FIG. 20.

In one embodiment, Compound I Form IX is characterized by a differentialscanning calorimetry (DSC) curve that comprises an endotherm at about152° C. In one embodiment, the DSC curve is substantially as shown inFIG. 21.

In one embodiment, Compound I Form IX is characterized by a weight loss,as measured by thermogravimetric analysis (TGA), of about 3.1% at about150° C. In one embodiment, the TGA is substantially as shown in FIG. 21.

Another embodiment is crystalline5-(3,3-dimethylbutyn-1-yl)-3-[(cis-4-hydroxy-4-{[(3S)-tetrahydrofuran-3-yloxy]methyl}cyclohexyl){[(1R)-4-methylcyclohex-3-en-1-yl]carbonyl}amino]thiophene-2-carboxylicacid methyl ethyl ketone (Compound I Form X), characterized by an X-raypowder diffractogram comprising at least three of the following peaks:6.9, 8.0, 15.7, 16.1, or 17.5 °2θ±0.2 °2θ, as determined on adiffractometer using Cu-Kα. In one embodiment, the diffractogramcomprises peaks at 6.9, 8.0, 15.7, 16.1, and 17.5 °2θ±0.2 °2θ. In oneembodiment, the diffractogram is substantially as shown in FIG. 22.

In one embodiment, Compound I Form X is characterized by a differentialscanning calorimetry (DSC) curve that comprises an endotherm at about150° C. In one embodiment, the DSC curve is substantially as shown inFIG. 23.

In one embodiment, Compound I Form X is characterized by a weight loss,as measured by thermogravimetric analysis (TGA), of about 3.1% at about150° C. In one embodiment, the TGA is substantially as shown in FIG. 23.

Another embodiment is crystalline5-(3,3-dimethylbutyn-1-yl)-3-[(cis-4-hydroxy-4-{[(3S)-tetrahydrofuran-3-yloxy]methyl}cyclohexyl){[(1R)-4-methylcyclohex-3-en-1-yl]carbonyl}amino]thiophene-2-carboxylicacid (Compound I Form XI), characterized by an X-ray powderdiffractogram comprising at least three of the following peaks: 5.4,5.7, 7.0, 7.9, or 8.6 °2θ±0.2 °2θ, as determined on a diffractometerusing Cu-Kα. In one embodiment, the diffractogram comprises peaks at5.4, 5.7, 7.0, 7.9, and 8.6 °2θ±0.2 °2θ. In one embodiment, thediffractogram is substantially as shown in FIG. 24.

In one embodiment, Compound I Form XI is characterized by a weight loss,as measured by thermogravimetric analysis (TGA), of about 3% at about160° C. In one embodiment, the TGA is substantially as shown in FIG. 25.

Another embodiment is crystalline5-(3,3-dimethylbutyn-1-yl)-3-[(cis-4-hydroxy-4-{[(3S)-tetrahydrofuran-3-yloxy]methyl}cyclohexyl){[(1R)-4-methylcyclohex-3-en-1-yl]carbonyl}amino]thiophene-2-carboxylicacid ethanol solvate (Compound I Form XII), characterized by an X-raypowder diffractogram comprising at least three of the following peaks:4.5, 6.4, 7.1, 8.5, or 8.9 °2θ±0.2 °2θ, as determined on adiffractometer using Cu-Kα. In one embodiment, the diffractogramcomprises peaks at 4.5, 6.4, 7.1, 8.5, and 8.9 °2θ±0.2 °2θ. In oneembodiment, the diffractogram is substantially as shown in FIG. 26.

In one embodiment, Compound I Form XII is characterized by a weightloss, as measured by thermogravimetric analysis (TGA), of about 4.9% atabout 100° C. In one embodiment, the TGA is substantially as shown inFIG. 27.

Another embodiment is amorphous5-(3,3-dimethylbutyn-1-yl)-3-[(cis-4-hydroxy-4-{[(3S)-tetrahydrofuran-3-yloxy]methyl}cyclohexyl){[(1R)-4-methylcyclohex-3-en-1-yl]carbonyl}amino]thiophene-2-carboxylicacid isopropyl alcohol solvate (amorphous Compound I). In oneembodiment, wherein the X-ray powder diffractogram as determined on adiffractometer using Cu-Kα, diffractogram is substantially as shown inFIG. 28.

Methods of Making Solid Forms of Compound I

One embodiment is a process for making Compound I Form I comprisingcontacting a5-(3,3-dimethylbutyn-1-yl)-3-[(cis-4-hydroxy-4-{[(3S)-tetrahydrofuran-3-yloxy]methyl}cyclohexyl){[(1R)-4-methylcyclohex-3-en-1-yl]carbonyl}amino]thiophene-2-carboxylicacid with water and a solvent selected from the group consisting ofacetonitrile, methanol, and acetone, whereby Compound I Form I isformed.

In one embodiment, the method further comprises isolating Compound IForm I.

One embodiment is a process for making Compound I Form II, comprisingcontacting5-(3,3-dimethylbutyn-1-yl)-3-[(cis-4-hydroxy-4-{[(3S)-tetrahydrofuran-3-yloxy]methyl}cyclohexyl){[(1R)-4-methylcyclohex-3-en-1-yl]carbonyl}amino]thiophene-2-carboxylicacid with acetonitrile, whereby Compound I Form II is formed.

In one embodiment, the method further comprises isolating Compound IForm II.

One embodiment is a process for making Compound I Form III, comprisingcontacting5-(3,3-dimethylbutyn-1-yl)-3-[(cis-4-hydroxy-4-{[(3S)-tetrahydrofuran-3-yloxy]methyl}cyclohexyl){[(1R)-4-methylcyclohex-3-en-1-yl]carbonyl}amino]thiophene-2-carboxylicacid with ethanol and water, whereby Compound I Form III is formed.

In one embodiment, the method further comprises isolating Compound IForm III.

One embodiment is a process for making Compound I Form IV, comprisingcontacting5-(3,3-dimethylbutyn-1-yl)-3-[(cis-4-hydroxy-4-{[(3S)-tetrahydrofuran-3-yloxy]methyl}cyclohexyl){[(1R)-4-methylcyclohex-3-en-1-yl]carbonyl}amino]thiophene-2-carboxylicacid with ethanol and water, whereby Compound I Form IV is formed.

In one embodiment, the method further comprises isolating Compound IForm IV.

One embodiment is a process for making Compound I Form V-MTBE, CompoundI Form V-IPA, Compound I Form V-EtOH, Compound I Form V-MEK, or CompoundI Form V-2-Me-THF, comprising contacting5-(3,3-dimethylbutyn-1-yl)-3-[(cis-4-hydroxy-4-{[(3S)-tetrahydrofuran-3-yloxy]methyl}cyclohexyl){[(1R)-4-methylcyclohex-3-en-1-yl]carbonyl}amino]thiophene-2-carboxylicacid with a solvent or solvent mixture selected from the groupconsisting of methyl tert-butyl ether, isopropyl alcohol and watermixture, ethanol and water mixture, methyl ethyl ketone and heptanemixture, and 2-methyl tetrahydrofuran and heptane mixture, wherebyCompound I Form V-MTBE, Compound I Form V-IPA, Compound I Form V-EtOH,Compound I Form V-MEK, or Compound I Form V-2-Me-THF, respectively, isformed.

In one embodiment, the method further comprises isolating Compound IForm V-MTBE, Compound I Form V-IPA, Compound I Form V-EtOH, Compound IForm V-MEK, or Compound I Form V-2-Me-THF.

One embodiment is a process for making Compound I Form VI, comprisingcontacting5-(3,3-dimethylbutyn-1-yl)-3-[(cis-4-hydroxy-4-{[(3S)-tetrahydrofuran-3-yloxy]methyl}cyclohexyl){[(1R)-4-methylcyclohex-3-en-1-yl]carbonyl}amino]thiophene-2-carboxylicacid with diisopropyl ether, whereby Compound I Form VI is formed.

In one embodiment, the method further comprises isolating Compound IForm VI.

One embodiment is a process for making Compound I Form VII, comprisingcontacting5-(3,3-dimethylbutyn-1-yl)-3-[(cis-4-hydroxy-4-{[(3S)-tetrahydrofuran-3-yloxy]methyl}cyclohexyl){[(1R)-4-methylcyclohex-3-en-1-yl]carbonyl}amino]thiophene-2-carboxylicacid with isopropyl alcohol, whereby Compound I Form VII is formed.

In one embodiment, the method further comprises isolating Compound IForm VII.

One embodiment is a process for making Compound I Form VIII, comprisingcontacting5-(3,3-dimethylbutyn-1-yl)-3-[(cis-4-hydroxy-4-{[(3S)-tetrahydrofuran-3-yloxy]methyl}cyclohexyl){[(1R)-4-methylcyclohex-3-en-1-yl]carbonyl}amino]thiophene-2-carboxylicacid with ethanol and water, whereby Compound I Form VIII is formed.

In one embodiment, the method further comprises isolating Compound IForm VIII.

One embodiment is a process for making Compound I Form IX, comprisingcontacting5-(3,3-dimethylbutyn-1-yl)-3-[(cis-4-hydroxy-4-{[(3S)-tetrahydrofuran-3-yloxy]methyl}cyclohexyl){[(1R)-4-methylcyclohex-3-en-1-yl]carbonyl}amino]thiophene-2-carboxylicacid with ethanol, whereby Compound I Form IX is formed.

In one embodiment, the method further comprises isolating Compound IForm IX.

One embodiment is a process for making Compound I Form X, comprisingcontacting5-(3,3-dimethylbutyn-1-yl)-3-[(cis-4-hydroxy-4-{[(3S)-tetrahydrofuran-3-yloxy]methyl}cyclohexyl){[(1R)-4-methylcyclohex-3-en-1-yl]carbonyl}amino]thiophene-2-carboxylicacid with methyl ethyl ketone, whereby Compound I Form X is formed.

In one embodiment, the method further comprises isolating Compound IForm X.

One embodiment is a process for making Compound I Form XI, comprisingdesolvating Compound I Form X, whereby Compound I Form XI is formed.

In one embodiment, the method further comprises isolating Compound IForm XI.

One embodiment is a process for making Compound I Form XII, comprisingcontacting Compound I Form I with ethanol in water, whereby Compound IForm XII is formed.

In one embodiment, the process further comprises isolating Compound IForm XII.

One embodiment is a process for making amorphous Compound I, comprisingcontacting Compound I Form II with dichloromethane, whereby amorphousCompound I is formed.

In one embodiment, the process further comprises isolating amorphousCompound I.

Pharmaceutical Compositions

The solid forms of Compound I provided in accordance with the presentdisclosure are usually administered in the form of pharmaceuticalcompositions. This disclosure therefore provides pharmaceuticalcompositions that contain, as the active ingredient, one or more of thesolid forms of Compound I described or a pharmaceutically acceptablesalt or ester thereof and one or more pharmaceutically acceptableexcipients, carriers, including inert solid diluents and fillers,diluents, including sterile aqueous solution and various organicsolvents, permeation enhancers, solubilizers and adjuvants. Thepharmaceutical compositions may be administered alone or in combinationwith other therapeutic agents (as indicated in the Combination Therapysection below). Such compositions are prepared in a manner well known inthe pharmaceutical art (see, e.g., Remington's Pharmaceutical Sciences,Mace Publishing Co., Philadelphia, Pa. 17th Ed. (1985); and ModernPharmaceutics, Marcel Dekker, Inc. 3rd Ed. (G. S. Banker & C. T. Rhodes,Eds.)

The pharmaceutical compositions may be administered in either single ormultiple doses by any of the accepted modes of administration of agentshaving similar utilities, for example as described in those patents andpatent applications incorporated by reference, including rectal, buccal,intranasal and transdermal routes, by intra-arterial injection,intravenously, intraperitoneally, parenterally, intramuscularly,subcutaneously orally, topically, as an inhalant or via an impregnatedor coated device such as a stent, for example or an artery-insertedcylindrical polymer.

One mode for administration is parenteral, particularly by injection.The forms in which the novel compositions of the present disclosure maybe incorporated for administration by injection include aqueous or oilsuspensions or emulsions, with sesame oil, corn oil, cottonseed oil orpeanut oil, as well as elixirs, mannitol, dextrose or a sterile aqueoussolution and similar pharmaceutical vehicles. Aqueous solutions insaline are also conventionally used for injection, but less preferred inthe context of the present disclosure. Ethanol, glycerol, propyleneglycol, liquid polyethylene glycol, and the like (and suitable mixturesthereof), cyclodextrin derivatives, and vegetable oils may also beemployed. The proper fluidity can be maintained, for example, by the useof a coating, such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.The prevention of the action of microorganisms can be brought about byvarious antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, thimerosal, and the like.

Sterile injectable solutions are prepared by incorporating a compoundaccording to the present disclosure in the required amount in theappropriate solvent with various other ingredients as enumerated above,as required, followed by filtered sterilization. Generally, dispersionsare prepared by incorporating the various sterilized active ingredientsinto a sterile vehicle which contains the basic dispersion medium andthe required other ingredients from those enumerated above. In the caseof sterile powders for the preparation of sterile injectable solutions,the general methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

Oral administration is another route for administration of compounds inaccordance with the disclosure. Administration may be via capsule orenteric coated tablets or the like. In making the pharmaceuticalcompositions that include at least one compound described herein, theactive ingredient is usually diluted by an excipient and/or enclosedwithin such a carrier that can be in the form of a capsule, sachet,paper or other container. When the excipient serves as a diluent, it canbe in the form of a solid, semi-solid or liquid material (as above),which acts as a vehicle, carrier or medium for the active ingredient.Thus, the compositions can be in the form of tablets, pills, powders,lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions,syrups, aerosols (as a solid or in a liquid medium), ointmentscontaining, for example, up to 10% by weight of the active compound,soft and hard gelatin capsules, sterile injectable solutions and sterilepackaged powders.

Some examples of suitable excipients include lactose, dextrose, sucrose,sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates,tragacanth, gelatin, calcium silicate, microcrystalline cellulose,polyvinylpyrrolidone, cellulose, sterile water, syrup and methylcellulose. The formulations can additionally include: lubricating agentssuch as talc, magnesium stearate and mineral oil; wetting agents;emulsifying and suspending agents; preserving agents such as methyl andpropylhydroxy-benzoates; sweetening agents; and flavoring agents.

The compositions of the disclosure can be formulated so as to providequick, sustained or delayed release of the active ingredient afteradministration to the patient by employing procedures known in the art.Controlled release drug delivery systems for oral administration includeosmotic pump systems and dissolutional systems containing polymer-coatedreservoirs or drug-polymer matrix formulations. Examples of controlledrelease systems are given in U.S. Pat. Nos. 3,845,770; 4,326,525;4,902514; and 5,616,345. Another formulation for use in the methods ofthe present disclosure employs transdermal delivery devices (“patches”).Such transdermal patches may be used to provide continuous ordiscontinuous infusion of the compounds of the present disclosure incontrolled amounts. The construction and use of transdermal patches forthe delivery of pharmaceutical agents is well known in the art. See,e.g., U.S. Pat. Nos. 5,023,252, 4,992,445 and 5,001,139. Such patchesmay be constructed for continuous, pulsatile or on demand delivery ofpharmaceutical agents.

In some embodiments, the compositions are formulated in a unit dosageform. The term “unit dosage forms” refers to physically discrete unitssuitable as unitary dosages for human subjects and other mammals, eachunit containing a predetermined quantity of active material calculatedto produce the desired therapeutic effect, in association with asuitable pharmaceutical excipient (e.g., a tablet, capsule, ampoule).The compounds are generally administered in a pharmaceutically effectiveamount. In some embodiments, each dosage unit contains from 1 mg to 2 gof a compound described herein and for parenteral administration, insome embodiments, from 0.1 to 700 mg of a compound a compound describedherein. It will be understood, however, that the amount of the compoundactually administered usually will be determined by a physician, in thelight of the relevant circumstances, including the condition to betreated, the chosen route of administration, the actual compoundadministered and its relative activity, the age, weight and response ofthe individual patient, the severity of the patient's symptoms, and thelike.

For preparing solid compositions such as tablets, the principal activeingredient is mixed with a pharmaceutical excipient to form a solidpreformulation composition containing a homogeneous mixture of acompound of the present disclosure. When referring to thesepreformulation compositions as homogeneous, it is meant that the activeingredient is dispersed evenly throughout the composition so that thecomposition may be readily subdivided into equally effective unit dosageforms such as tablets, pills and capsules.

The tablets or pills of the present disclosure may be coated orotherwise compounded to provide a dosage form affording the advantage ofprolonged action or to protect from the acid conditions of the stomach.For example, the tablet or pill can comprise an inner dosage and anouter dosage component, the latter being in the form of an envelope overthe former. The two components can be separated by an enteric layer thatserves to resist disintegration in the stomach and permit the innercomponent to pass intact into the duodenum or to be delayed in release.A variety of materials can be used for such enteric layers or coatings,such materials including a number of polymeric acids and mixtures ofpolymeric acids with such materials as shellac, cetyl alcohol andcellulose acetate.

Compositions for inhalation or insufflation include solutions andsuspensions in pharmaceutically acceptable, aqueous or organic solventsor mixtures thereof and powders. The liquid or solid compositions maycontain suitable pharmaceutically acceptable excipients as describedsupra. In some embodiments, the compositions are administered by theoral or nasal respiratory route for local or systemic effect.Compositions in preferably pharmaceutically acceptable solvents may benebulized by use of inert gases. Nebulized solutions may be inhaleddirectly from the nebulizing device or the nebulizing device may beattached to a facemask tent or intermittent positive pressure breathingmachine. Solution, suspension or powder compositions may beadministered, in some embodiments orally or nasally, from devices thatdeliver the formulation in an appropriate manner.

In one embodiment, this disclosure relates to a pharmaceuticalcomposition comprising a pharmaceutically acceptable excipient orcarrier and a therapeutically effective amount of the compound ofFormula I as described above or a pharmaceutically acceptable salt,ester, prodrug, stereoisomer or hydrate thereof.

Methods of Use

The solid forms of Compound I described herein are administered to asubject suffering from hepatitis C virus (HCV) in either single ormultiple doses by any of the accepted modes of administration known tothose who are skilled in the art and as detailed above.

Combination Therapy

Subjects being treated by administration of Compound I forms describedin the present disclosure can benefit from treatment with additionaltherapeutic agents that are effective in treating HCV, or enhance theanti-HCV therapeutic effect of Compound I forms, in accordance with someembodiments. Additional therapeutic agents that are useful for thispurpose include, but are not limited to, ribavirin,

The present disclosure is not to be limited in scope by the specificembodiments disclosed in the examples, which are intended to beillustrations of a few embodiments of the disclosure, nor is thedisclosure to be limited by any embodiments that are functionallyequivalent within the scope of this disclosure. Indeed, variousmodifications of the disclosure in addition to those shown and describedherein will become apparent to those skilled in the art and are intendedto fall within the scope of the appended claims. To this end, it shouldbe noted that one or more hydrogen atoms or methyl groups can be omittedfrom the drawn structures consistent with accepted shorthand notation ofsuch organic compounds, and that one skilled in the art of organicchemistry would readily appreciate their presence.

EXAMPLES Example 1 Method of Making5-(3,3-dimethylbutyn-1-yl)-3-[(cis-4-hydroxy-4-{[(3S)-tetrahydrofuran-3-yloxy]methyl}cyclohexyl){[(1R)-4-methylcyclohex-3-en-1-yl]carbonyl}amino]thiophene-2-carboxylicacid

I. Synthesis of Starting Materials

A. Epoxidation, Etherification, Deketalization, Reductive Amination, andHydrogenolysis to Provide Intermediate 6

1. Epoxidation to Prepare Intermediate 2

Charge lithium tert-butoxide (1.14 kg, 1.1 equiv) andtrimethylsulfoxonium iodide (3.12 kg, 1.1 equiv) to an inerted 70 Lreactor with the jacket temperature set to 23° C. Charge DMSO (13.8 kg)and vigorously mix contents between 20 and 25° C. for one hour. Charge1,4-cyclohexanedione monoethylene acetal (2.02 kg, 1.0 equiv) to thereactor. Once reaction is complete, charge the reactor with brine (18 L,15 wt %) at a rate to ensure the reaction temperature does not exceed40° C. Extract the homogenous brine containing reaction mixture withMTBE (3×30 kg) and combine the product containing organics. Concentratethe combined organics by distillation at ambient pressure. Distill offthe MTBE to 5 volumes (10 L) to provide a solution of 2 in MTBE.

¹H NMR (400 MHz, CDCl₃) δ 4.02-3.91 (m, 4H), 2.67 (s, 2H), 1.95-1.83 (m,4H), 1.81-1.72 (m, 2H), 1.60-1.53 (m, 2H).

2. Etherification to Prepare Intermediate 3

Charge the solution of 2 in MTBE, (S)-tetrahydrofuran-3-ol (1.25 kg, 1.1equiv), and potassium tert-butoxide (1.59 kg, 1.1 equiv) to an inerted70 L reactor and heat contents to 55 to 60° C. Upon reaction completion,cool the reactor contents to ambient to afford a solution of 3 in MTBE.

¹H NMR (400 MHz, CDCl₃) δ 4.19-4.09 (m, 1H), 4.00-3.66 (m, 8H), 3.27(dd, J=20.1, 8.8 Hz, 2H), 2.04-1.84 (m, 6H), 1.76-1.68 (m, 2H),1.67-1.50 (m, 2H).

3. Deketalization to Prepare Intermediate 4

Charge HCl (13.3 L, 1.5 N) to the reactor containing a solution of 3 inMTBE (2.4 kg, 11.1 mmol). Mix the biphasic reaction mixture between 18and 25° C. Once the reaction is complete, stop agitation and separatethe two phases. Extract the aqueous phase with CH₂Cl₂ (2×L). Combineorganic phases in the reactor. Charge aqueous NaHCO₃ (15 L, 7.5 wt %)and mix for 1 hour, let settle and split phases. Return organic phase toreactor. Concentrate organics to 5 vol (10 L). Charge 12 L of ethanoland concentrate to 8.5 L to afford 4 as a solution in EtOH. ¹H NMR (400MHz, CDCl₃) δ 4.17 (m, 1H), 3.95-3.75 (m, 4H), 3.35 (AB, 2H), 2.74 (td,J=13.6, 6.7 Hz, 2H), 2.10-1.97 (m, 5H), 1.71 (td, J=13.6, 6.7 Hz, 2H).

4. Reductive Amination to Prepare Intermediate 5

Charge the EtOH solution of 4 to the 70 L reactor (7.84 kg, 30.3 wt %,by ¹H-NMR), and set the jacket temperature to 20° C. Charge Ti(OiPr)₄(4.0 kg, 1.25 equiv) at a pace to keep the reaction temperature below30° C. Charge benzylamine (1.2 kg, 1.0 equiv) at a rate to keep thereaction temperature below 35° C. Agitate the reaction mixture for 1hour at 20° C., then cool the reaction mixture to −4° C. Dissolve sodiumborohydride (210 g, 0.5 equiv) in EtOH (5.8 L), and charge to thereaction mixture at a rate to maintain a reaction temperature of notmore than 0° C. After 1 hour, add 20 wt % trisodium citrate solution (38L) and 4-methylpentan-2-one (MIBK, 19.2 L) and set the jackettemperature to 20° C. Agitate the mixture vigorously for 30 minutes, andsettle layers. Discard the aqueous layer, and wash the organic phasewith 15 wt % NaCl (19 L). Concentrate the organic phase under reducedpressure to an oil. Charge MIBK (7.1 L, 3 vol) and polish filter theturbid solution through a 0.6 micron filter. Transfer the filtrate tothe 70 L reactor and charge (S)-mandelic acid (1.7 kg, 1.0 equiv)followed by a small amount of seed crystal. Age the slurry for at least1 hour, and then add MTBE (9.6 L, 4 vol) over 15 minutes. Filter theslurry and wash the cake with 8.7 L 2/1 MIBK/MTBE. Dry the solid invacuum oven to afford 5 as white solid.

¹HNMR (400 MHz, CD₃OD) δ 7.60-7.38 (m, 7H), 7.32-7.18 (m, 3H), 4.21-4.09(m, 3H), 3.89-3.71 (m, 4H), 3.34-3.19 (m, 2H), 3.12-2.97 (m, 1H), 2.34(d, J=7.1 Hz, 1H), 1.96 (ddd, J=46.9, 24.1, 3.7 Hz, 2H), 1.85-1.65 (m,4H), 1.51 (td, J=13.7, 3.7 Hz, 2H).

5. Hydrogenolysis to Prepare Intermediate 6

Charge 5 (2.20 kg, 1 equiv) and Pd(OH)₂/C (0.12 kg, 20 wt % dry basis,0.02 equiv) to a 70 L reactor. Inert the reactor and charge MeOH (25.8L, 10 vol) and ammonium formate (1.52 kg, 5.0 equiv). Warm the reactorcontents to 48-50° C. under positive nitrogen pressure and agitate. Whenthe reaction is complete, cool the reaction mixture to 18 to 25° C., andfilter the reaction mixture to remove the solids. Solvent exchangethrough distillation under reduced pressure to isopropanol (IPA)targeting a final volume of 12 L (5 vol). Filter the slurry and wash thecake with IPA (4 L, 2 vol). Dry the solid in vacuum oven at 40° C. toobtain 6 as white solid.

¹H NMR (400 MHz, CD₃OD) δ 7.50-7.42 (m, 2H), 7.33-7.15 (m, 3H),4.15-4.06 (m, 1H), 3.92-3.59 (m, 4H), 3.37-3.13 (m, 4H), 2.94 (td,J=10.7, 5.6 Hz, 1H), 1.97 (td, J=7.7, 4.4 Hz, 2H), 1.87-1.59 (m, 7H),1.55-1.39 (m, 2H).

B. Bromination, De-Bromination, and Alkynylation to Provide Intermediate9

1. Bromination to Prepare Intermediate 7

A reactor was charged with a solution of aqueous hydrobromic acid(47.6%, 125.0 kg, 5 equiv). Thiophene (12.5 kg, purity 99%, 1 equiv) wasadded to it at 25-30° C. Tetrabutylammonium bromide (0.625 kg, 0.13equiv) was added to the reaction mass. The reaction mass was heated to50-55° C. 50% Aqueous hydrogen peroxide solution (31.3 kg, 3.1 equiv)was added to the reaction mass over 10 h keeping the temperature in therange of 50-55° C. The reaction mass was then heated to 70-75° C. Afterreaction completion, the reaction mass was cooled to 20-25° C. andwashed with 20% sodium metabisulphite solution (17 L), 2 N sodiumhydroxide solution (62 L) and the crude product was subjected tofractional distillation using a 2 ft wire-mesh packed column to afford2,3,5-tribromothiophene. The spectral properties of this molecule areconsistent with commercially available material.

2. De-Bromination to Prepare Intermediate 8

Dimethyl sulfoxide (DMSO, 330 L) was charged to reactor.2,3,5-tribromothiophene (33 kg, 1.0 equiv) was charged to the reactionmass under stirring. The reaction mass was cooled to 15-20° C. Sodiumborohydride (7.8 kg, 2.0 equiv) was charged lot wise to the reactionmass in 2.0 h maintaining temperature 15 to 20° C. The reaction mass washeated to 20 to 25° C. and maintained until the reaction was completed.The reaction mass was quenched in water (660 L) at 10 to 15° C. and theproduct was extracted into toluene (5×165 L). The combined organic layerwas washed with water (165 L). The organic layer was dried overanhydrous sodium sulfate (8.0 kg) and concentrated under reducedpressure below 50° C. to yield 2,4 dibromothiophene. The spectralproperties of this molecule are consistent with commercially availablematerial.

3. Alkynylation to Provide Intermediate 9

A reactor was evacuated and flushed with nitrogen. Dimethyl formamide(560 L) and 2,4-dibromothiophene (37.5 kg, 1.0 equiv) were charged tothe reactor. The reaction mass was cooled to 20-25° C. Palladiumchloride bis-triphenylphosphine complex (3 kg, 0.03 equiv) was chargedto the reaction mass followed cuprous iodide (1.6 kg, 0.06 equiv),t-butyl acetylene (13.0 kg, 1.1 equiv) and triethylamine (43 kg, 3.0equiv). The reactor was again flushed with nitrogen and pressurized with0.50 kg nitrogen (extra pure) pressure. The reaction mass was heated to25-30° C. and agitated until reaction completion (ca. 6 h). The reactionmass was filtered and the filter cake was washed with dimethyl formamide(37.5 L). The filtrate was concentrated under reduced pressure at atemperature below 50° C. The residue was dissolved in heptane (187.5 L)at 25 to 30° C. The solids were filtered off and washed with heptane(3×56 L). The filtrate was washed successively with 5% ammonia solutionand saturated NaCl solution. The organic layer was further dried overanhydrous sodium sulfate and concentrated under vacuum at a temperaturebelow 70° C. The crude oil was purified by fractional distillation toprovide 9.

¹H NMR (400 MHz, CDCl₃) δ 7.00 (s, 1H), 6.96 (s, 1H), 1.26 (s, 9H).

C. Diels-Alder and Saponification to Provide Intermediate 13

Charge a reactor with (S)-diphenylprolinol (1.26 kg, 0.0625 equiv) andtri-o-tolylboroxine (0.59 kg, 0.0213 equiv) and toluene (40 L).Concentrate the reactor contents at atmospheric pressure until anapproximate volume of 10 L. Cool the reaction mixture to 0° C. andcharge a solution of triflimide (1.11 kg, 0.05 equiv) in anhydrous DCM(7.3 L) at a rate to ensure the reaction temperature does not exceed 10°C. Charge 2,2,2-trifluoroethyl acrylate (12.2 kg, 1.0 equiv) at a rateto ensure the temperature does not exceed 10° C. Cool the mixture to 0°C. and charge isoprene (8.05 kg, 2.0 equiv) slowly over approximately 4h maintaining a reaction temperature of 0° C. Upon reaction completion,concentrate the reaction mixture until the content of DCM is less than20% relative to the intermediate ester. Charge tetrahydrofuran (THF, 69L) and heat the solution to 40° C. Charge a solution lithium hydroxidemonohydrate (LiOH.H₂O, 4.0 kg, 1.2 equiv) in 46 L of water over 1 h andstir until the saponification reaction is complete as determined by TLC.Concentrate the reaction mixture until less than 20 mol % THF remainsrelative to 13 by NMR. Charge methyl tert-butyl ether (MTBE, 50 L) andwash with water (6.1 L). Back extract the aqueous layer with MTBE (2×50L). Discard the combined organics and concentrate product containingaqueous phase until than 5 mol % MTBE remains relative to 13. To theaqueous mixture charge heptane (46 L) and DCM (2.4 L). Wash the biphasicmixture with 4 M HCl (31 kg). Back extract the aqueous layer withn-heptane (52 L) and wash combined organics with 0.1M HCl (15 kg), and20% brine (38 kg). To the organic solution of 13 in DCM/heptane chargemorpholine (6.9 kg, 1.05 equiv) over 2 h at 20° C. Filter the resultingslurry and wash the filter cake with n-heptane (36 L). Drying the solidsunder vacuum at 35° C. provides 13 (98.4% ee).

¹H NMR (400 MHz, CDCl₃) δ 7.76 (s, 2H), 5.39-5.37 (m, 1H), 3.77-3.74 (m,4H), 2.98-2.96 (m, 4H), 2.48-2.41 (m, 1H), 2.27-2.12 (m, 2H), 2.10-1.90(m, 3H), 1.74-1.62 (m, 1H), 1.65 (s, 3H).

II. Synthesis of Compound I

A. N-Arylation, acylation, and carboxylation to provide Compound I

1. N-Arylation to Prepare Formula III

Charge a 125-mL reaction vessel with Pd₂(dba)₃ (57 mg, 0.3 mol %),t-Bu-BippyPhos (0.63 g, 7 mol %), and KOH (3.5 g, 3.0 equiv). Inert thevessel and charge t-amylalcohol (40 mL, 8 vol), water (2 mL, 0.4 vol), 6(9.1 g, 1.2 equiv), and 9 (5.0 g, 1.0 equiv). Inert vessel and heatreactor contents to 90° C. until the reaction is complete as determinedby the consumption of 9. Cool reaction mixture to 23° C. and concentratemixture under reduced pressure to give brown solids. Purify crude solidsby silica gel chromatography in EtOAc to provide III as a tan solidhaving 99.9:0.1 diastereomeric ratio by achiral HPLC.

¹H NMR (400 MHz, CDCl₃) δ 6.60 (d, J=2.0 Hz, 1H), 5.78 (d, J=2.0 Hz,1H), 4.16-4.10 (m, 1H), 3.88 (dd, J=16.6, 7.9 Hz, 1H), 3.84-3.76 (m,3H), 3.25 (dd, J=19.2, 8.7 Hz, 2H), 3.09-2.98 (m, 1H), 2.01-1.95 (m,2H), 1.94-1.90 (m, 2H), 1.77-1.74 (m, 2H), 1.59-1.44 (m, 2H), 1.42-1.31(m, 2H), 1.29-1.27 (m, 9H).

2. Acylation to Provide Formula IV

A 15-mL flask equipped with a magnetic stir bar and a nitrogen inlet wascharged with 13 (557 mg, 1.5 equiv), 2-methyl-THF (5 mL) and a drop ofDMF (ca. 2 μL). The reaction mixture was cooled to 4° C. using an icebath. To the reaction mixture was added oxalyl chloride (0.32 mL, 1.4equiv) dropwise over 1 min. The reaction mixture was allowed to warm to19° C. over 30 min and aged at 19° C. for 3 h. To a 50-mL flask equippedwith a magnetic stir bar and a nitrogen inlet were added III (1.00 g,1.0 equiv), MeTHF (5 mL) and diisopropylethylamine (1.38 mL, 3 equiv),and the contents were cooled to 7° C. using an ice bath. To the slurryof III was added the solution of the acid chloride dropwise over 5 min.The reaction mixture was allowed to warm to 17° C. over 30 min and agedfor 3 h. The reaction mixture was quenched with 10 wt % aqueous citricacid (10 mL) and the phases were separated. The organic phase was washedwith water (10 mL) and concentrated under reduced pressure. The residuewas dissolved in isopropanol (25 mL) and concentrated to ca. 5 mL. Tothe solution was added water (5 mL) over 10 min and seed crystal of IV(5 mg, 0.5 wt %). The slurry was aged at room temperature for 16 h andfiltered. The filter cake was rinsed with 1/1 IPA/water (6 mL) and driedin a vacuum oven for 24 h to afford IV.

¹H NMR (400 MHz, CDCl₃) δ 6.86 (d, J=1.8 Hz, 1H), 6.82 (d, J=1.8 Hz,1H), 5.30-5.25 (m, 1H), 4.55-4.45 (m, 1H), 4.15-4.03 (m, 2H), 3.89-3.72(m, 4H), 3.32-3.20 (m, 2H), 2.30-2.18 (m, 2H), 1.99-1.67 (m, 11H),1.56-1.36 (m, 4H), 1.34-1.30 (m, 9H).

3. Carboxylation to Provide Compound I

In a 50 mL flask, IV (1.00 g, 1.0 equiv) and THF (10 mL) were placed,and the solution was cooled to −11° C. using acetone/ice bath. To thesolution was added n-BuLi (2.4 mL, 2.5 M solution in hexanes, 3 equiv)over 10 min maintaining internal temperature below −3° C. The reactionmixture was allowed to age between −12° C. to −10° C. for 1 h. Carbondioxide (lecture bottle equipped with a pressure regulator) wasintroduced through a needle, and the bubbling was continued for 10 min.The reaction mixture was aged at −10° C. for 1 h, quenched with 10 wt %aqueous citric acid (10 mL) and allowed to warm to 19° C. The layerswere separated and the organic solution was diluted with isopropylacetate (50 mL). The solution was concentrated to ca. 5 mL under reducedpressure. To the solution was then added benzylamine (0.22 mL, 1 equiv).The slurry was aged for 30 min, and filtered. The filter cake was rinsedwith iPAc (10 mL). In a 50 mL flask were added the wet cake of I, iPAc(10 mL) and 10 wt % aqueous citric acid (10 mL). The mixture was stirreduntil all solids dissolved, and the phases were separated. The organicphase was washed with water (10 mL), and diluted with iPAc (50 mL). Tothe slurry was added heptane (10 mL) over 2 h, and the slurry wasfiltered. The filter cake was washed with 2/1 heptane/iPAc (6 mL) anddried in vacuum oven. Note: Slow interconversion between two rotamers onNMR timescale gives rise to two sets of NMR signals.

¹H NMR (400 MHz, DMSO-d6) δ 13.48 (br. s., 1H), 7.21, s; 7.16, s, (1H),5.28, m; 5.24, m, (1H); 4.32 (m, 1H); 4.06 (m, 1H); 3.99 (br. s, 1H);3.70 (dd, J=8.0, 15.2 Hz, 1H); 3.65 (ddd J=8.0, 15.2, 3.2 Hz, 1H); 3.63(m, 2H), 3.10 (dd, J=9.6, 1.6 Hz, 1H), 3.06 (d, J=9.6 Hz, 1H), 2.20, m;2.09, m, (1H); 2.05, m; 1.90, m, (1H); 1.86 (m, 2H); 1.86, m; 1.82, m,(1H); 1.80, m; 1.76, m, (1H); 1.70, m; 1.64, m (1H); 1.68, m; 1.63, m(1H); 1.55, m; 1.38, m (1H); 1.54, m; 1.42, m (2H); 1.52 (s, 3H); 1.48,m; 1.16, m (2H); 1.46 (m, 2H); 1.42 (m, 2H); 1.30, s, 1.29, s (9H).

Example 2 Form Screening

I. Stable Form Screening

A. Initial Stable Form Screening

The first stable form screening was conducted using 19 solvents/mixturesat ambient conditions. The starting material was amorphous.Anti-solvents, either water or heptane, were used to precipitate thesolids. The results are summarized in Table 1.

TABLE 1 Results from the first stable form screening for Compound IInit. Conc. 24 h 2-Week Solubility Solvent (mg/mL) solids solids (mg/mL)Anti-solvent Solids Water 11 amorphous amorphous ND — — ACN 91.6 II II13 — — MeOH 87.4 solution solution >87.4 water I EtOH 76 solutionsolution >76 water V Acetone 79.6 solution solution >79.6 water I IPA95.4 solution solution >95.4 water V MEK 65.6 solution solution >65.6heptane V MIBK 60.2 solution solution >60.2 heptane II MeCl₂ 36 solutionsolution >36 heptane II THF 53.4 solution solution >53.4 water + heptaneI 2 Me-THF 77.4 solution solution >77.4 heptane V EtOAc 49.4 solutionsolution >49.4 heptane II IPAc 39.6 solution solution >39.6 heptane I +II MTBE >45 solution V 316 — — Toluene 16.2 solution solution >16.2heptane II Heptane 12.7 amorphous amorphous ND — — EtOH/water 26 III IV14.5 — — ACN/water 45.8 I I 0.23 — — IPA/water 47.7 solution V 1.78 — —

After 24 h, three crystalline forms were observed in the slurry. Form Iis a hydrate form obtained from ACN/water 50:50 mixture. Form II is ananhydrous form obtained from ACN. Form III is a hydrate/solvate formfrom ethanol/water mixture, and it converts to Form IV upon drying.Solids in water and heptane were amorphous. Other samples remained assolutions.

After 2 weeks, Form V was obtained in MTBE and IPA/water mixture. Itappears to be a solvated form. Additionally, Form III converted to FormIV in the slurry. Solids in water and heptane remained amorphous. Othersamples remained as solution.

When anti-solvents (water or heptane) were added to the remainingsolutions, all solutions crystallized. No new forms appeared based onXRPD.

B. Follow-Up Stable Form Screening

In the follow-up stable form screening experiments, the startingmaterials were Form II. The solids were stirring in the solvents listbelow at about 22° C. in 2 ml vials. Results in Table 2 showed that FormII was stable in most tested solvents except for alcohols, water, andMEK.

TABLE 2 Second stable form screening results Solubility Solvent 24 hForm 2-Week Form (mg/mL) Water II + I (small amt) I 0.25 (I) EtOH/water(52/48) V V 8 (V) ACN II II 6 MeOH solution solution >321 EtOH IX IX 283(IX) Acetone II II 241 IPA VII VII 266 (VII) MEK X X 127 (X) MIBK II II116 DCM solution solution >274 THF solution solution >346 2 Me—THFsolution solution >334 Ethyl Acetate II II 124 Isopropyl Acetate II II59 Methyl t-butyl ether II II 108 Toluene II II 40 Heptane II II 0.05ACN/water (50/50) II II 18

C. Stable Form Competition Study

To further verify the stability of Form II, competitive crystallizationwas conducted by adding Forms I through V into a high concentrationsolution of amorphous Compound I in ACN. After stirring overnight, FormII was the only detected form. The mother liquor obtained, which wassaturated in terms of Form II, was used in another test in which otherforms (Forms I, III, IV and V) were added. Those solids all dissolvedquickly, again confirming that Form II was more stable in ACN.

II. Hydrate Screening

A. ACN/Water System

ACN does not form solvates with Compound I, so it is appropriate to useACN and water mixture in this study. The results are shown in Table 3.

TABLE 3 Hydrate screen using ACN/water Water Water Equivalent StartingForm at Form at 2 content activity RH (%) Form 24 h or 90 h weeks  0% 00 II II (24 h) II 4.5%  0.6 60 II II (24 h) II 6.6%  0.7 70 II II (24 h)II 10% 0.779 77.9 II II (90 h) II 11.5%  0.8 80 II II (24 h) II 20%0.863 86.3 II II (90 h) II 30% 0.896 89.6 II II (90 h) VIII 32% 0.9 90II II (24 h) VIII 40% 0.914 91.4 II II (90 h) VIII 50% 0.926 92.6 IIII + small amount VIII VIII (90 h) 60% 0.936 93.6 II VIII + small amountVIII II (90 h) 70% 0.943 94.3 II II (90 h) VIII 80% 0.949 94.9 II II (90h) II + VIII 80.9%  0.950 95 II I + II (24 h) I 90% 0.955 95.5 II II (90h) I 100%  1 100 II I + II (24 h) I

In this study, Form II was shown to be stable in up to 0.863 wateractivity (or 86.3% RH) at 22° C. In higher water activity conditions,neither Form VIII or Form I is more stable but the conversion rate isgenerally slow.

B. EtOH/Water System

When the organic component used in the solvent mixture forms solvatewith Compound I, potential hydrates may not show up in the slurryscreen, as shown in the hydrate screening using ethanol/water mixtures(Table 4). Form IX is a new form found in this study.

TABLE 4 Hydrate screen using ethanol/water Water Water Starting FormForm content activity Form at 90 hours at 2 weeks  0% 0 II IX IX 10%0.510 II solution solution 20% 0.654 II solution solution 30% 0.721 II VV 40% 0.770 II V V 50% 0.806 II III + small V amount V 60% 0.838 II IIIIV 70% 0.866 II III IV 80% 0.893 II I I 90% 0.921 II I I 100%  1 II I I

C. Form II Stability in Water at Elevated Temperature

During formulation, Compound I may be wet granulated and dried at <50°C. A slurry of Form II in water was stirred at 48° C. (50° C. jacket)and was sampled periodically for XRPD analysis to determine Form Icontent. Based on FIG. 6, Form I was not detected at the 6 hour point;the sample slowly converted to Form I; and after 30 hours, Form I becamethe dominant form in the XRPD. The XRPD for Form I is as shown in FIG.1.

III. Desolvation Screening

Another method of generating anhydrous forms is desolvation. In thisstudy, the solvates and hydrates of Compound I were heated to about110˜120° C. in a vacuum oven to desolvate. The results are shown inTable 5.

TABLE 5 Desolvation of different forms TGA desolvation Form Solvent TempOven Temp Final Form I Water  40-70° C. 110~120° C. Amorphous VEtOH/water  50-140° C. 110~120° C. Amorphous (52/48) VI DIPE 100-150° C.110~120° C. VI VII IPA 100-130° C. 110~120° C. Amorphous VIII Water/ACN 50-80° C. 110~120° C. VIII (60:40) IX EtOH 100-150° C. 110~120° C. IX XMEK  50-125° C. 110~120° C. XI

In this study, Form X changed to Form XI upon heating.

IV. Summary of the Crystalline Solids of Compound I

Table 6 summarizes the crystalline forms observed in the stable formscreenings, hydrate screening, and desolvation screening. Form XII,which was found later, is also included in this table.

TABLE 6 Comparison of the various forms of Compound I Melting Classifi-point Weight loss Forms cation Compatible solvents (° C.) by TGA (%) Ihydrate Water, ACN/water, 123 5.4 MeOH/water, acetone/ water, THF/waterII anhydrous ACN, MIBK, MIBK/ 161.6 0.2 heptane, toluene, DCM/heptane,EtOAc, iPrOAc, MTBE III hydrate/ EtOH/water 125 4.4 solvate IV hydrate/EtOH/water 125 2.8 solvate V solvate MTBE, IPA/water, 123~131 3.5~4.8EtOH/water, MEK/heptane, 2Me—THF/heptane VI solvate DIPE 144 4.6 VIIsolvate IPA 115 6.9 VIII hydrate ACN/water 119 3.1 IX solvate EtOH 1463.2 X solvate MEK 137 3.1 XI anhydrous (generated by desol- 140~150 3.0vation of Form X) XII solvate EtOH/water Not 4.9 determined

Example 3 Characterization of Form II

I. XRPD

The XRPD pattern for this form and the other forms described herein wasobtained in the following experimental setting: 45 KV, 45 mA, Kα1=1.5406Å, scan range 2. 40°, step size 0.0084°, counting time: 8.25 s. The XRPDpattern of Form II is shown in FIG. 4. Its major characteristic peaksare 5.5, 6.0, 9.6, 9.9, 16.9, 18.3, 21.3° 2θ.

The peaks and relative intensities of the peaks in the XRPD for Form IIare provided in Table 7 below.

TABLE 7 Relative intensities of peaks for XRPD of Form II No. Position[°2 θ] Relative Intensity [%] 1 5.5 100 2 6.0 62 3 9.6 7 4 9.9 9 5 12.15 6 13.1 6 7 14.5 2 8 16.5 8 9 16.9 25 10 18.1 19 11 19.2 9 12 19.4 1513 19.8 16 14 20.1 6 15 20.8 10 16 21.3 24 17 21.5 5 18 21.9 3 19 25.3 220 26.8 2 21 31.2 2 22 36.4 1II. TGA and DSC

The TGA and DSC data of Form II are plotted in FIG. 5. Form II isanhydrous and lost 0.2% mass when heated to about 120° C. It has amelting point of about 161.6° C., the highest among all forms discoveredso far.

Example 4 Preparation of Form II

The procedure of making Form II is as follows:

A solution of 10 g of Compound I and 70 mL of isopropyl acetate iswarmed to 40° C. and optionally seeded with 50 mg Form II seed. Theresulting thin slurry is agitated for at least 1 h at 40° C. and then140 mL heptane was added dropwise over 3 h. The slurry was optionallycooled to 0-5° C. over 2 h and continued to stir at least 2 h. Thesolids were isolated by filtration and the wet cake was rinsed with 20mL heptane, and then dried in a vacuum oven at 40° C.

Example 5 Preparation and Characterization of Additional Forms

Form I

Form I is a hydrate and was obtained from water and varioussolvent/water mixtures such as ACN/water, MeOH/water, acetone/water,THF/water. FIG. 3 compares the XRPD patterns of Form I obtained fromACN/water, MeOH/water, acetone/water mixtures. They closely resembleeach other.

TGA data are plotted in FIG. 2 of Form I solids. The solids obtainedfrom water (by converting from Form II), ACN/water, MeOH/water, andacetone/water mixtures have similar TGA behavior, and they lost about5.4% water upon heating. Karl Fischer analysis showed that the solidsfrom ACN/water contain about 5.18% water, which is consistent with theTGA data. The exact stoichiometry of water in the crystal lattice isunknown because 5.4% is between sesquihydrate (4.7%) and lower thandihydrate (6.2%).

Loss of water during heating caused the crystal lattice to collapse, asconfirmed by XRPD patterns at 50 and 70° C. in FIG. 7. The solidspartially became amorphous after drying at 70° C.

Forms III and IV

Form III and Form IV emerged in EtOH/water mixture at water contents of60 to 70% (Table 4). Experiments showed that Form III converted to FormIV upon drying or in slurry after 2 weeks of stirring. Form III (FIG. 8)and Form IV (FIG. 10) have similar XRPD patterns except for shifting ofseveral peaks. TGA data (shown in FIG. 9 and FIG. 11) indicate that theyare both hydrate/solvate forms.

Form V

Form V was obtained from MTBE, EtOH/water, IPA/water, MEK/heptane,2-Me-THF/heptane, and their XRPDs are shown in FIG. 12.

Form V appears to be a solvate form, as shown in FIG. 13 for solids fromEtOH/water mixture. The TGA weight losses and melting points are not thesame for solids from different solvents, as shown in Table 8. Thesesolids from different solvent systems could be isostructural solvates.

TABLE 8 Comparison of the TGA weight loss and melting temperatures ofForm V from different solvents TGA Weight loss (%) Melting temperature(° C.) MTBE 4.8 123.6 IPA/water 4.2 Not determined EtOH/water 3.5 130.7MEK/heptane Not determined 124.9 2Me—THF/heptane Not determined 129.1Form VI

Form VI was detected in a sample crystallized from diisopropyl ether(DIPE). The XRPD, TGA and DSC data are shown in FIG. 14 and FIG. 15,respectively. It is a DIPE solvate and lost about 4.7% weight at about150° C.

In an attempt crystallize Form VI, the solution of Compound I in DIPEwas seeded with Form VI. However, only Form II was obtained. Thisexperiment shows that Form II is more stable than Form VI.

Form VII

Form VII appeared in the follow-up stable form screening using IPA assolvent (Table 2). Its XRPD pattern is shown in FIG. 16 and its DSC andTGA data are shown in FIG. 17. This form is a solvate of IPA based onthe weight loss of about 6.9% at about 140° C.

Form VIII

Form VIII was obtained in ACN/water system at water contents of 30 to80% (Table 3). It appears to be a monohydrate according to the weightloss of about 3.1%. The XRPD is shown in FIG. 18 and the DSC and TGA areshown in FIG. 19.

Form IX

Form IX is a solvate of ethanol that appeared in the follow-up stableform screen (Table 2). TGA shows that it is a solvate. The XRPD patternand TGA data are shown in FIG. 20 and FIG. 21.

Form X

Form X emerged in the follow-up stable form screen (Table 2) using MEK.TGA shows that it is a solvate. The XRPD pattern, TGA and DSC data areshown in FIG. 22 and FIG. 23.

Form XI

Form XI is crystalline solids that formed when Form X (MEK solvate) washeated to about 110˜120° C. (Table 5). The XRPD pattern is shown in FIG.24.

When analyzed by TGA, the sample shows significant weight loss uponheating before reaching about 110° C. (FIG. 25). This was probablycaused by absorption of moisture during sample transfer.

Form XII

Form XII was observed in an experiment in which water was adding to FormIX slurries in ethanol in an attempt further precipitate the solute.After stirring overnight, Form XII was the only solids present in theslurry. Its XRD pattern is shown in FIG. 26. It appeared to be asolvate/hydrate as shown by TGA data in FIG. 27.

Amorphous

Amorphous Compound was provided by dissolving 1 g of Form II in 3 mLdichloromethane at ambient temperature. The solution was filtered, andthe solvent evaporated at about 30° C. and under vacuum. The solids werefurther dried in a vacuum oven at 40° C. for 3 h. The XRPD pattern isshown in FIG. 28.

We claim:
 1. Crystalline5-(3,3-dimethylbutyn-1-yl)-3-[(cis-4-hydroxy-4-{[(3S)-tetrahydrofuran-3-yloxy]methyl}cyclohexyl){[(1R)-4-methylcyclohex-3-en-1-yl]carbonyl}amino]thiophene-2-carboxylicacid hydrate (Compound I Form I) characterized by an X-ray powderdiffractogram comprising at least three of the following peaks: 4.0,7.3, 8.1, 17.9, or 21.8° 2θ±0.2° 2θ, as determined on a diffractometerusing Cu-Kα radiation.
 2. Compound I Form I of claim 1, wherein thediffractogram comprises peaks at 4.0, 7.3, 8.1, 17.9, and 21.8° 2θ±0.2°2θ.
 3. Compound I Form I of claim 1, wherein the diffractogram issubstantially as shown in FIG.
 1. 4. Compound I Form I of claim 1,characterized by a differential scanning calorimetry (DSC) curve thatcomprises two endotherms at about 55° C. to about 115° C. and about 127°C.
 5. Compound I Form I of claim 4, wherein the DSC curve issubstantially as shown in FIG.
 2. 6. Compound I Form I of claim 1,characterized by a weight loss, as measured by thermogravimetricanalysis (TGA), of about 5.4% at about 95° C.
 7. Compound I Form I ofclaim 6, wherein the TGA is substantially as shown in FIG.
 2. 8.Crystalline5-(3,3-dimethylbutyn-1-yl)-3-[(cis-4-hydroxy-4-{[(3S)-tetrahydrofuran-3-yloxy]methyl}cyclohexyl){[(1R)-4-methylcyclohex-3-en-1-yl]carbonyl}amino]thiophene-2-carboxylicacid (Compound I Form II) characterized by an X-ray powder diffractogramcomprising at least three of the following peaks: 5.5, 6.0, 16.9, 18.1,or 21.3° 2θ±0.2° 2θ, as determined on a diffractometer using Cu-Kα. 9.Compound I Form II of claim 8, wherein the diffractogram comprises peaksat 5.5, 6.0, 16.9, 18.1, and 21.3° 2θ±0.2° 2θ.
 10. Compound I Form II ofclaim 8, wherein the diffractogram is substantially as shown in FIG. 4.11. Compound I Form II of claim 8, characterized by a differentialscanning calorimetry (DSC) curve that comprises an endotherm at about162° C.
 12. Compound I Form II of claim 11, wherein the DSC curve issubstantially as shown in FIG.
 5. 13. Compound I Form II of claim 8,characterized by a weight loss, as measured by thermogravimetricanalysis (TGA), of about 0.2% at about 120° C.
 14. Compound I Form II ofclaim 13, wherein the TGA is substantially as shown in FIG.
 5. 15. Amethod for treating a subject suffering from hepatitis C virus (HCV),comprising administering to the subject a therapeutically effectiveamount of a compound of claim
 1. 16. The method of claim 15, comprisingfurther administering to the subject at least one anti-HCV agent. 17.The method of claim 16, wherein the anti-HCV agent is a compoundselected from the group consisting of ribavirin,


18. A pharmaceutical composition comprising the compound of claim 1 anda pharmaceutically acceptable excipient.
 19. A process for makingCompound I Form I of claim 1, comprising contacting5-(3,3-dimethylbutyn-1-yl)-3-[(cis-4-hydroxy-4-{[(3S)-tetrahydrofuran-3-yloxy]methyl}cyclohexyl){[(1R)-4-methylcyclohex-3-en-1-yl]carbonyl}amino]thiophene-2-carboxylicacid with water and a solvent selected from the group consisting ofacetonitrile, methanol, and acetone, whereby Compound I Form I isformed.
 20. The process of claim 19, further comprising isolatingCompound I Form I.
 21. A process for making Compound I Form II of claim8, comprising contacting5-(3,3-dimethylbutyn-1-yl)-3-[(cis-4-hydroxy-4-{[(3S)-tetrahydrofuran-3-yloxy]methyl}cyclohexyl){[(1R)-4-methylcyclohex-3-en-1-yl]carbonyl}amino]thiophene-2-carboxylicacid with acetonitrile, whereby Compound I Form II is formed.
 22. Theprocess of claim 21, further comprising isolating Compound I Form II.23. A method for treating a subject suffering from hepatitis C virus(HCV), comprising administering to the subject a therapeuticallyeffective amount of Compound I Form II of claim
 8. 24. The method ofclaim 23, comprising further administering to the subject at least oneanti-HCV agent.
 25. The method of claim 24, wherein the anti-HCV agentis a compound selected from the group consisting of ribavirin,


26. A pharmaceutical composition comprising the compound of claim 8 anda pharmaceutically acceptable excipient.